WO2000023620A1 - Procede de production d'empreintes genetiques - Google Patents

Procede de production d'empreintes genetiques Download PDF

Info

Publication number
WO2000023620A1
WO2000023620A1 PCT/NL1999/000643 NL9900643W WO0023620A1 WO 2000023620 A1 WO2000023620 A1 WO 2000023620A1 NL 9900643 W NL9900643 W NL 9900643W WO 0023620 A1 WO0023620 A1 WO 0023620A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna
fragments
primer
amplification
aflp
Prior art date
Application number
PCT/NL1999/000643
Other languages
English (en)
Inventor
Henricus Maria Verbakel
Hubertus Mechtilda Petrus Segers
Michael Josephus Theresia Van Eijk
Johannes Petrus Schouten
Yin-Lay Chan
Original Assignee
Keygene N.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Keygene N.V. filed Critical Keygene N.V.
Priority to AU63720/99A priority Critical patent/AU6372099A/en
Publication of WO2000023620A1 publication Critical patent/WO2000023620A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • the present invention relates to nucleic acid amplification methods, nucleic acid detection methods and probes for the detection of nucleic acids.
  • the invention more specifically relates to methods for the generation of DNA fingerprints for the identification and typing of nucleic acid in a sample.
  • the DNA genome of living organisms (except viruses) consists of a sequence of 500.000 - 5.000.000.000 or more building blocks (bases) . In order to detect (small) differences between different genomes, a limited fraction of this complex DNA has to be highlighted.
  • DNA fingerprints In case no sequence information of the genome is known, a usual starting point for the generation of DNA fingerprints is the fragmentation of the DNA preparation in smaller parts by bacterial enzymes (restriction-endonucleases) . Subsequently a limited number of the DNA fragments obtained is highlighted, either by hybridisation with a labelled probe or by amplification of a subset of the fragments, e.g. with the use of the polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • PCR has been used extensively during the last years to amplify simultaneously different fragments of the chromosomal DNA from a organism, in order to obtain a "fingerprint" of the particular genome. In these cases it is often not important which fragments are amplified, provided that the reaction is completely reproducible and the number of fragments that are amplified are roughly controllable.
  • DNA fingerprints are used to show similarities and differences between DNA samples obtained from different individuals, races or species.
  • Some of the techniques used are RAPD ( Random Amplified Polymorphic DNA) which suffers from insufficient reproducibility between different laboratories and AFLP ( Amplified Fragment Length Polymorphism, Vos et al 1995) .
  • the AFLP technique involves four steps : 1) Fragmentation of the DNA by restriction-endonucleases. 2) Ligation of adapters to the ends of the DNA fragments. 3) Amplification of a subset of the fragments by a set of carefully selected oligonucleotides, one of which is labelled, e.g. with a fluorescent tag. 4) Visualisation of the amplified fragments by gel-electrophoresis and a suitable detection method. For fragmentation of the genome in a reproducible way, the AFLP method uses restriction-endonucleases that generate a single stranded tail of usually 2 - 4 bases on each fragment.
  • a small DNA fragment (adapter) can be attached to these single strands, provided that the adapter also has a single stranded part and that the two single stranded parts are complementary each other.
  • These adapters have a known DNA sequence. Apart from the adapter sequence, also the sequence recognised by the restriction enzyme is known. This information can be used to design primers for the amplification of all fragments starting with an adapter and followed by the restriction endonuclease site. In order to limit the number of fragments amplified, some random bases can be added to the 3 ' end of one or both PCR primers (selective bases) .
  • Adding a first selective nucleotide results in a primer that is not completely complementary anymore to the adapter and/or adjacent restriction enzyme site. Instead, depending on the code of the first nucleotide following the restriction enzyme site, the primer will have a mismatch in three of the four fragments. Thus the number of fragments preferentially amplified is reduced by four. For each additional selective base added to the three prime end of the primer, the number of fragments preferentially amplified with this primer decreases by a further factor of four.
  • a drawback of the AFLP method is that it usually requires two consecutive cycles of PCR for the analysis of complex genomes.
  • the technique is also rather sensitive to changes in protocol, preventing the replacement of the expensive Mse 1 enzyme by other restriction endonucleases .
  • Another drawback of the AFLP technique as it is currently used is the sensitivity to minor impurities in the DNA preparations.
  • Another drawback of the AFLP technique is that the number of bands obtained with complex genomes often cannot be limited to less than approx. 30 as the number of selective bases used cannot be increased to more than three without the risk of extra bands due to mismatching of misalignment between PCR primers and template. For certain applications a further reduction in the number of bands is desirable.
  • the present invention has proven to be much less sensitive to changes in protocol and to impurities in the DNA preparations.
  • at least one restriction endonuclease more is used than in the AFLP technique, the number of suitable enzymes is large permitting the choice of cheap and stable enzymes .
  • the DNA-PARTIAL ADAPTER LIGATION-SELECTIVE AMPLIFICATION invention discussed below, the number of efficiently amplified DNA fragments is reduced 20-fold compared to the AFLP technique, which limits the number of selective nucleotides needed on the primers used and permits a further reduction of the number of bands amplified. BRIEF DESCRIPTION OF THE DRAWINGS.
  • Figure 1 shows a graphic outline of the general selective amplification mechanism of the DNA-PARTIAL ADAPTER LIGATION- SELECTIVE AMPLIFICATION invention.
  • Figure 2 shows a 6 % polyacrylamide gel with DNA fingerprints obtained by applying the DNA-PARTIAL ADAPTER LIGATION- SELECTIVE AMPLIFICATION invention to E. ccli genomic DNA.
  • Figure 3 shows a 6 % polyacrylamide gel with DNA fingerprints obtained by applying the DNA-PARTIAL ADAPTER LIGATION- SELECTIVE AMPLIFICATION invention to different Cyncta individuals .
  • Figure 4 shows a 6 % polyacrylamide gel with DNA fingerprints obtained by applying the DNA-PARTIAL ADAPTER LIGATION- SELECTIVE AMPLIFICATION invention to different Lycopersicum strains .
  • Figure 5 shows a 6 % polyacrylamide gel with a DNA fingerprint obtained by applying the DNA-PARTIAL ADAPTER LIGATION-SELECTIVE AMPLIFICATION invention to human genomic DNA.
  • Figure 6 shows a detail of Figure 5.
  • Figure 7 Two possible ways to amplify part of the sequence of an individual AFLP marker using an internal amplification primer PI or P2.
  • the AFLP marker is an EcoRI +2/ Msel +3 AFLP marker but the general principle applies to other restriction enzyme combinations and numbers of selective nucleotides in the AFLP primer (s) .
  • EcoRI +2 refers to an AFLP primer with 2 selective bases
  • Msel +3 refers to an Msel AFLP primer with three selective bases
  • internal PI refers to an internal PCR primer in forward direction
  • internal P2 refers :o an internal PCR primer in the reverse direction
  • Figure 8 Schematic presentation of a resul- of an AFLP sequence tagging. DETAILED DESCRIPTION OF THE DNA-PARTIAL ADAPTER LIGATION- SELECTIVE AMPLIFICATION INVENTION. Definitions
  • restriction endonucleases and “restriction enzymes” refer to bacterial enzymes each of which cut double-stranded DNA at or near a specific nucleotide sequence.
  • oligonucleotide and “oligomer” are defined as molecules comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than three.
  • One or more of the nucleotides can be modified e.g. by addition of a methyl group, a biotin or digoxigenin moiety, a fluorescent tag or by using radioactive nucleotides.
  • the term "primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of nucleic acid sequence synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, i.e. in the presence of four different nucleotide triphosphates and a polymerase in an appropriate buffer ( "buffer” includes pH, ionic strength, cofactors etc.) and at a suitable temperature.
  • buffer includes pH, ionic strength, cofactors etc.
  • One or more of the nucleotides of the primer can be modified e.g. by addition of a methyl group, a biotin or digoxigenin moiety, a fluorescent tag or by using radioactive nucleotides.
  • DNA polymorphism refers to the condition in which two or more different nucleotide sequences can exist at a particular site in the DNA.
  • nucleotide variation in sequence refers to any type of nucleotide variation being caused either by single or multiple nucleotide substitutions, or by deletions or insertions. These nucleotide variations may be mutant or polymorphic allele variations.
  • RFLP restriction fragment length polymorphism
  • PCR refers to the polymerase chain reaction (Mulis et al U.S.Pat.Nos. 4,683,195, 4,683,202 and 4,800,159).
  • the PCR amplification process results in the exponential increase of discrete DNA fragments whose length is defined by the 5 ' ends of the oligonucleotide primers .
  • DNA fragments from a complex mixture of fragments has been published (Vos et al , 1985 ; European patent application no. 534 858 Al) .
  • This method known as amplified fragment length polymorphism (AFLP) , is based on the digestion of a DNA preparation with two restriction-endonucleases, followed by the ligation of DNA oligomers to the ends of these fragments.
  • a subset of the resulting fragments are amplified by the Polymerase Chain Reaction (PCR) , separated by gel- electrophoresis, and visualised by a suitable detection method, e.g. autoradiography .
  • PCR Polymerase Chain Reaction
  • a DNA mixture is obtained with three different kinds of fragments : 1) Fragments having two ends generated by the same restriction-enzyme and having type A adapters attached to both ends; 2) Fragments having two B-type adapters and 3) Fragments having one A and one B type adapter.
  • the number of fragments amplified is reduced in three ways : First, the two primers complementary to the adapters differ in their annealing temperature. The primer with the higher annealing temperature is preferentially used during a PCR reaction favouring the amplification of fragments with at least one adapter complementary to this primer.
  • the restriction enzymes for the digestion of the DNA are chosen in a way that a vast majority of the fragments contain adapters that are both complementary to the primer with the lowest annealing temperature and will thus be amplified less efficiently.
  • the fragments having two different adapters attached are preferentially amplified, as the ends of fragments having two identical adapters are able to anneal to each other as well as to the PCR primers.
  • the third way to limit the number of fragments amplified is the inclusion of selective bases in the primers.
  • Fragments need to have an adapter attached and thus have ends consisting of an adapter sequence and a restriction endonuclease recognition site, but also need to have a specific sequence of 1 - 3 bases next to the restriction endonuclease recognition site in order to be amplified.
  • the AFLP method as described is to some extent sensitive to changes in the protocol, such as a change in restriction enzymes used, and impurities in the DNA preparations. These drawbacks of the AFLP method are due to the fact that all DNA-fragments contain adapter sequences at both ends. Each DNA fragment thus can act as a template during the polymerase chain reaction and all fragments produced during a PCR cycle will be a template during the next PCR cycle, as all fragments produced have an adapter sequence complementary to one of the PCR primers. Due to competition during the PCR cycles, the fragments having two different adapters attached are enriched using the described protocol .
  • the fragments containing one EcoRI and one Mse 1 adapter will be amplified by the Polymerase Chain Reaction more efficiently than the other fragments.
  • the amplification step of the AFLP technique should be considered as a competition in which only the carefully selected conditions will give successful results.
  • the number of fragments amplified is usually too large for analysis. The number of different fragments amplified is therefore limited by the addition of up to three selective bases to each of the primers. Theoretically the number of amplified fragments is reduced by a factor 4 for every selective base added to one of the primers .
  • a process for the analysis of nucleic acids comprising providing said nucleic acids in a double stranded DNA form wherein said process further comprises fragmenting said DNA to produce a collection of DNA fragments with at least three different types of ends.
  • at least two types of oligomer adapters are ligated to at least two different types of ends.
  • At least a first oligomer primer complementary to the first adapter and a second oligomer complementary to the second adapter are provided and said DNA fragments are amplified, whereby at least one type of DNA end does not take part in the amplification reaction.
  • DNA ends not taking part in the amplification reaction are meant those DNA ends from which no amplification is initiated in the reaction. Since a DNA fragment of the invention has two DNA ends, amplification of the DNA end not taking part in the amplification reaction may be initiated from the opposing DNA end. However, since in this case the amplification is not exponential, said fragment will not be a predominant amplification product and not contribute significantly to the DNA-fingerprint .
  • a suitable but non- limiting method to prevent a DNA end from taking part in the amplification reaction is not to supply a primer complementary to said DNA end in the amplification reaction. Fragmentation of the DNA is preferably accomplished by means of one or more restriction enzyme digestions. However, other DNA fragmenting enzymes may also be used for the present invention as long as they produce specific fragments and do not cut apparently at random such as a DNase .
  • the DNA ends taking part in the amplification reaction are fewer in number than the DNA ends not taking part in the amplification reaction.
  • the fragmenting of DNA is achieved with three restriction enzymes, two of which preferably recognise a 6 bp sequence and the third preferably recognises a 4 bp sequence, wherein preferably the enzyme recognising a 4 bp sequence is used to produce the DNA fragment end that is not taking part in the amplification reaction.
  • the fragmenting of the DNA occurs simultaneously to the ligation of the adapters.
  • one primer has an essentially lower annealing temperature than the another primer.
  • At least one primer is labelled, preferably the primer with the lowest annealing temperature. Labelling may occur through any suitable method such as through biotin, digoxigenin, fluorescent tags or radioactivity.
  • the labelled primer is labelled at its 5 'end.
  • at least one primer further comprises one or more selective nucleotides, leading to the preferred amplification of a subset of all DNA fragments containing the adapter to which the primer was complementary.
  • mRNA in a sample is converted into double stranded DNA, using methods known in the art such as reverse transcription, prior to performing the method of the invention.
  • the resulting DNA fingerprint is, in this case, in effect a fingerprint of the mRNA in a sample.
  • a kit useful for performing the method of the invention wherein the kit minimally comprises at least one method for fragmenting DNA to produce a collection of DNA fragments with at least three different types of ends.
  • the amplification of the fragments is achieved by means of polymerase chain reaction.
  • the invention is further illustrated using this amplification method.
  • the invention is not limited to amplification through polymerase chain reaction.
  • the NASBA amplification reaction or other methods may also be used.
  • only a fraction of the DNA fragments generated in the DNA fragmenting step are further processed in the method of the invention, i.e. a fraction of the fragments is purified from the pool of fragments generated. Said purification may occur through any means applicable.
  • specific purification oligomers can be generated that comprise of a sequence complementary to the sequence of interest and further comprise a means of purification, such as a biotin tag or attachment to an immobile substrate.
  • one of the primers added is not complementary to the known sequence of the adapters but instead complementary to a different known sequence, resulting in the specific amplification of only those fragments containing the known sequence.
  • This specific embodiment may be useful in, for instance but not limited to, the characterisation of retroviral or transposon integration sites.
  • the primer not complementary to the adapter sequences is complementary to a retroviral or transposon sequence.
  • AFLP sequence tagging Another preferred embodiment where one of the primers added is not complementary to the known sequence of the adapters but instead complementary to a different known sequence, resulting in the specific amplification of only those fragments containing the known sequence is termed AFLP sequence tagging.
  • the process termed AFLP * sequence tagging amplifies a single AFLP marker from an AFLP template or pre- amplification reactions, with the aim to develop a dominant PCR test for this AFLP marker.
  • This method is based on the use of an AFLP primer, in combination with an oligonucleotide primer that is derived from the internal sequence of the AFLP marker.
  • AFLP sequence tagging can be used to obtain an essentially specific signal (one or a few fragments) for the relevant polymorphism (s) , essentially without the large number of fragments resulting from "standard” AFLP. Thereby enabling an easier detection of said polymorphism.
  • the necessity of having at least one primer present comprising a specificity for an adapter is probably best exemplified in the following non-limiting example.
  • information for the specific polymorphism lies in the adapter ligated to the exact cutting site plus eventual selective bases in the primer directed to said adapter. To amplify this information one may use a further primer directed toward an adapter ligated to the other end of the fragment.
  • a specific primer is used, directed against a known sequence in the fragment, thereby eliminating a large number of essentially irrelevant fragments .
  • the primer directed toward the adapter comprising the polymorphic information cannot be replaced by a specific primer.
  • the AFLP sequence tagging method of the invention is now further exemplified below. The method is based on the use of an AFLP primer, in combination with a oligonucleotide primer that is derived from the internal sequence of the AFLP marker.
  • AFLP templates are prepared using two restriction enzyme combinations (followed by adapter ligation)
  • AFLP marker in an AFLP fingerprint is the result of a polymorphism in at least one restriction enzyme site and/or the adjacent selective nucleotides, there are different expected outcomes of the use of the two primer combinations EcoRI+2- internal P2 or Msel +3 - internal PI, depending on the molecular polymorphism that is responsible for the particular AFLP marker.
  • both primer combinations (EcoRI+2- internal P2 or Msel +3 - internal PI) will result in a PCR product, when taking AFLP templates or preamplification reactions as starting material.
  • PCR product formation with the two primer combinations depends on the position of the AFLP marker polymorphism in the EcoRI restriction enzyme site, Msel restriction enzyme site, a selective base on the Msel site and/or a selective base on the EcoRI site. In at least one of these positions a polymorphism is present resulting in the absence of the AFLP markers in the fingerprint of the individual.
  • EcoRI+2- P2 or Msel +3 -PI this means that:
  • PCR product formation starting from +0/+0 , +1/+0 or +2/+0 AFLP preamplification reactions is dependent on the position of the nearest Msel site in the genome. For relatively small AFLP markers there is a fair chance that the nearest Msel site is located within amplifiable distance in which case PCR product formation occurs with primer combination EcoRI+2 - internal P2. In this case the PCR product will have a different length.
  • PCR product when there is only polymorphism in one of the selective nucleotides on the EcoRI side, PCR product is expected with primer combination MseI+3 - internal PI, starting from AFLP templates, +0/+0 and +0/+3 preamplification reactions but not when starting from +2/+0 preamplification reactions.
  • primer combination EcoRI+0 - internal P2 is dependent upon the position of the polymorphism at the + 1 or +2 selective EcoRI base, but PCR product will be formed with when AFLP templates, +0/+0 and +0/+3 preamplifications are used as the starting material.
  • PCR product when there is only polymorphism located at one of the selective nucleotides on the Msel side, PCR product is expected with primer combination EcoRI - internal P2 when using AFLP templates or +0/+0 and +2/+0 preamplification reactions as starting material but not when using +0/+3 preamplifications .
  • primer combination MseI+0 - internal PI is dependent on the position of the polymorphism at the +l,+2 or +3 selective Msel base, but PCR product is expected when using AFLP templates and +0/+0 preamplification reactions and possibly also +2/+1 or +2/+2 (pre) amplification reactions as starting material.
  • PCR product refers to specific PCR product that is derived from the AFLP marker that is targeted with the internal PI or internal P2 primer.
  • Non- limited applications of AFLP sequence tagging include the development of a dominant PCR assay for markers of interest (such as AFLP markers with predictive value for (a) trait (s) of interest, amplification of AFLP fragments derived from gene-families members, targeting of AFLP markers containing specific conserved domain sequences, such as those present in certain types of plant disease resistance genes, and targeting of retrotransposon containing restriction fragments .
  • markers of interest such as AFLP markers with predictive value for (a) trait (s) of interest
  • amplification of AFLP fragments derived from gene-families members targeting of AFLP markers containing specific conserved domain sequences, such as those present in certain types of plant disease resistance genes, and targeting of retrotransposon containing restriction fragments .
  • the number of amplified fragments is limited by digesting the DNA with two restriction enzymes generating different ends, and ligating different adapters to these ends, as well as digesting the DNA with one or more other fragmenting enzymes for which no adapters are added.
  • the fragmenting enzymes are restriction enzymes.
  • the ends for which no specific adapters are added are fragmented by enzymes that cut the DNA (much) more often than the two restriction- enzymes that are used to produce the ends for which adapters are added.
  • 4 different kinds of fragments are produced: 1) Fragments without any adapter sequence (the majority of fragments in the preferred embodiment) . These will not be amplified at all. 2) Fragments having one adapter sequence. These may be amplified but not in an exponential way, as the fragments produced will have no sequence complementary to one of the PCR primers. 3) Fragments having two identical adapters and 4) Fragments having two different adapters. These latter two kinds of fragments will be exponentially amplified as with the usual AFLP protocol; In a preferred embodiment these two types of fragments are present in roughly equimolar amounts.
  • the fragments with two different adapters are preferentially amplified.
  • the annealing temperature of the primers is chosen such that the primer with the lower annealing temperature is labelled (In contrast to the AFLP method in which the labelled primer has the highest annealing temperature.).
  • Fragments containing two unlabeled primers may be amplified but are not detected. Amplification of fragments with two labelled primers is not efficient as the annealing temperature of these primers is low. Amplification of fragments with one labelled and one unlabeled primer is strongly favoured.
  • the mixture of DNA fragments is obtained by digesting the DNA with a mixture of 3 restriction- endonucleases . Two of these enzymes recognise a 6 bp sequence and the third enzyme recognises a 4 bp sequence.
  • the adapters are ligated to all ends made by digestion with the two 6 bp cutters. The digestion and the ligation reaction may be performed simultaneously. Part of the digestion-ligation reaction is used as a template in an amplification reaction such as PCR.
  • the amplification primers used are complementary to the adapters used in the digestion- ligation step but may contain selective nucleotides in order to reduce the number of fragments amplified.
  • the results can be visualised e.g. by gel-electrophoresis .
  • the resulting gel-patterns ("fingerprints") can be used e.g. to distinguish closely related organisms such as different strains of one organism.
  • A, B and C Restriction- endonucleases: A, B and C, having a, b and c cutting sites.
  • the number of fragments obtained is a + b + c.
  • the number of DNA-ends is thus 2 (a+b+c) .
  • 6 different kinds of fragments are generated.
  • the fraction of fragments with 2 ends both generated by the A-enzyme is a 2 / (a+b+c) 2
  • the fraction of fragments with 2 ends both generated by the B-enzyme is b2/ (a+b+c) 2
  • the fraction of fragments with 2 ends both generated by the C-enzyme is c2/ (a+b+c) 2
  • the fraction of fragments having one end generated by the A- enzyme and one end generated by the B-enzyme is 2ab/ (a+b+c) 2.
  • the fraction of fragments having one end generated by the A- enzyme and one end generated by the C-enzyme is 2ac/ (a+b+c) 2.
  • the fraction of fragments having one end generated by the B- enzyme and one end generated by the C-enzyme is 2bc/ (a+b+c) 2.
  • the fraction of fragments that can be amplified is b2+c 2+2bc/ (a+b+c) 2.
  • the total number of fragments that can be amplified is b2+c 2+2bc/ (a+b+c) .
  • the fraction of the fragments that are amplified efficiently is 2bc/ (a+b+c) 2.
  • the total number of fragments that can be efficiently amplified is 2bc/ (a+b+c)
  • DNA-partial adapter ligation-selective amplification using two enzymes with a 6 bp recognition site and one enzyme with a 4 bp recognition site The B- and C-enzymes (both with an 6 bp recognition site) have approximately the same number of cutting sites.
  • Ban 2 instead of two enzymes with a 6 bp recognition site, an enzyme such as Ban 2 can be used. Ban 2 has several different 6 bp recognition sites to which different adapters can be ligated.
  • DNA-partial adapter ligation-selective amplification using two enzymes with a 7 bp recognition site and one enzyme with a 4 bp recognition site The B- and C-enzymes (both with an 7 bp recognition site) have approximately the same number of cutting sites.
  • an enzyme such as Rsr 2 can be used, having two different 7 bp recognition sites to which two different adapters can be ligated.
  • the number of fragments that are potentially amplified can thus be easily reduced 80 - 1000 fold compared to the AFLP method (100 % vs . 1, 23 % or 0.1 %) .
  • the number of fragments that are efficiently amplified is easily reduced 20 - 220 fold (11 % vs. 1,23 % or 0.05 %) .
  • using two restriction enzymes recognising 6 bases for producing the ends taking part in the amplification and using a restriction enzyme recognising 4 bases for producing the ends not taking part in the amplification reaction only approx.
  • 1 % of the fragments can be amplified exponentially, compared to 100 % of the fragments in the AFLP method.
  • the approx. 0.6 % of the fragments that have two different adapters has to compete only with the 0.6 % of the fragments that have two identical adapters.
  • the present invention it is possible to use longer primers and higher annealing temperatures during the PCR cycles, resulting in more stringent amplifications and lower background .
  • the A enzyme for which no adapters are added cuts 15.625 times (on average every 256 bp) .
  • the B and C enzymes for which adapters are added each cut 976 times ( every 4096 bp . ) .
  • the total number of fragments that can be amplified will be 216. OF these, 108 will be amplified efficiently as they have two different adapters attached to their ends. For bacterial genomes, it will therefore not be necessary to further reduce the number of fragments by using selective bases on the amplification primers in case the fragments are separated on a long acrylamide gel . Addition of one selective nucleotide to each of the PCR primers will further reduce the average number of fragments amplified to 7, permitting the use of agarose gels with ethidium bromide for separation and detection.
  • the AFLP method will generate on average almost 2000 fragments on a bacterial 4.000.000 bp genome (without selective nucleotides) and on average 400 fragments on a human genome when using three selective nucleotides on each PCR primer.
  • restriction sequences may be substantially underrepresented. It is therefore often possible to select for a certain organism restriction endonucleases which recognise 6 bp and which cleave on average less than once every 4096 bp. In the AFLP method the number of fragments is reduced 10 fold when the enzyme used cleaves only once every 40960 bp. In the three restriction enzyme embodiment of the invention, using two enzymes that cleave on average only once every 40960 bp . the number of fragments amplified is reduced 100-fold.
  • DNA-PARTIAL ADAPTER LIGATION-SELECTIVE AMPLIFICATION method is much more robust than the AFLP technique described by Zabeau and Vos .
  • complex DNA e.g. human or plant DNA
  • DNA-PARTIAL ADAPTER LIGATION-SELECTIVE AMPLIFICATION fingerprints on these DNA's using a combined digestion-ligation reaction of 1 - 2 hr, followed by a single PCR reaction.
  • a typical DNA-PARTIAL ADAPTER LIGATION-SELECTIVE AMPLIFICATION fingerprint is obtained in less time, and at reduced costs compared to a typical AFLP fingerprint.
  • Dicrestion-licration reaction No change in pattern was observed when DNA samples of between 1 and 1000 ng tomato DNA / 20 ul reaction were used. The pattern changed when only 0.2 ng DNA / 20 ul was used. As only 0.5 ul of the digestion/ligation reaction was used for the PCR reaction, this corresponds to 5 pg tomato DNA / PCR reaction, or only approx. 5 haploid genome equivalents. Using 40 ng tomato DNA / 20 ul digestion/ligation reaction, no change in pattern was observed when reaction times were reduced to 30 minutes or extended to 4 hours, or if the incubation temperature was raised to 37 °C or lowered to 25 °C (at an 1.5 hr incubation time) .
  • restriction endonucleases Although specific restriction endonucleases are recited in the Examples, it will be recognised that isoschizomers, i.e. enzymes that have the same recognition se ⁇ uence, or other restriction enzymes can be substituted and the same or equivalent results will be achieved.
  • isoschizomers i.e. enzymes that have the same recognition se ⁇ uence, or other restriction enzymes can be substituted and the same or equivalent results will be achieved.
  • the following examples illustrate but do not limit the invention.
  • Enzyme-mixtures used contain 1 unit/ul of each restriction- endonuclease + 0.25 Weiss units T4-DNA-ligase .
  • 1 unit restriction-endonuclease as the amount of enzyme needed to digest to completion in 1 hr at 30 °C, 1 ug of a hypothetical DNA with 1 site in every 4096 bp . for each enzyme with a 6 bp recognition site and 1 site in every 256 bp. for each enzyme with a 4 bp . recognition site.
  • the buffer used contains 10 mM Tris-HCl pK 7,6 ; 50 mM NaCl ; 6 mM MgCl2 and 0.5 mM ATP. Activity of enzymes was measured with unmethylated Lambda DNA as substrate and was afterwards corrected for site density in Lambda DNA.
  • unit for the activity of T4-DNA-ligase is defined as the amount of enzyme able to convert 1 nmol (32P) from pyrophosphate into Norit-absorbable material in 20 min at 37 °C (Weiss units) .
  • Fun 2 is an isoschizomer of EcoRI, but has no star activity and has a higher activity at temperatures lower than 37 °C.
  • SpaHl is an isoschizomer of Sph 1. EXAMPLE 2 .
  • This example pertains to the generation of a DNA fingerprint of DNA isolated from phages Lambda, T7 and Adenovirus.
  • the size of the fragments amplified with the DNA-PARTIAL ADAPTER LIGATION-SELECTIVE AMPLIFICATION method can be predicted.
  • Genbank accession numbers J02459 (Lambda) , V01146 (T7) and J01917 (Adenovirus) were used.
  • This mix was added to a digestion/ligation reaction in a final volume of 20 ul containing 10 mM Tris-HCl pH 7,6 ; 50 mM NaCl ; 10 mM MgCl2 ; 1 mM DTT ; 2 pMol of each of four oligomers (SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6), 0.5 unit T4-DNA ligase ; 0.5 unit Rsa 1 ; 0.5 unit Fun 2 ; 0.5 unit SpaHl. Incubation was performed for 1.5 hr at 30 °C.
  • the 50 ul PCR reaction contained 1 ul of the digestion/ligation reaction as template, as well as 10 pMol of an 5--FITC labelled primer complementary to the Fun 2 adapter and having one selective nucleotide on its 3 ' end (T ; SEQ ID NO 7) , 10 pMol of an unlabeled primer complementary to the SpaHl adapter (no selective nucleotides, SEQ ID NO 8) , 15 mM Tris-HCl pH 8.5 , 50 mM KCl , 1.5 mM MgCl2 , 0.01 % nonionic detergent such as Triton X-100 , 200 uM of each of the four dNTP's and 0.5 unit Taq polymerase.
  • the PCR mixture was overlayered with mineral oil. Following the PCR reaction, 8 ul of this reaction was analysed on a 1.8 % agarose gel in 0.5 x TBE buffer. The results obtained were strong bands on a agarose gel with lengths of approx. 1500, 480 and 270 bp.
  • This example pertains to the generation of DNA fingerprints of genomic DNA isolated from an E. coli strain, lysogenic for phage Lambda .
  • Genomic DNA was isolated from E. coli strain DM1 (Life Technologies Inc.).
  • SpaHl is an isoschizomer of Sph 1.
  • Adapters Eco (SEQ ID NO 3 and SEQ ID NO 4) + Sph ( SEQ ID NO 5 and SEQ ID NO 6) .
  • Primers FITC-labelled Eco-T (SEQ ID NO: 7) and unlabeled Sph-0 (SEQ ID NO: 8) Lane 2: As in lane 1, but Dde I was replaced by Mbo I. Lane 3: Fun 2, Bel I and Rsa I.
  • Adapters Eco (SEQ ID NO 3 and SEQ ID NO 4) + Bam ( SEQ ID NO 9 and SEQ ID NO 10) .
  • the Lambda fragment between nucleotides 39168 and 39423 of the Lambda sequence is the only Lambda fragment flanked by EcoRI and Sph 1 sites that does not contain a Dde 1 or Mbo I site and that contains a T nucleotide next to the EcoRI site.
  • the 254 bp fragment will generate a FITC labelled 284 nucleotide fragment.
  • This example pertains to the generation of a DNA fingerprint from different specimens of a small insect (Orchesella cincta ; springtails) .
  • DNA from different individuals was purified by phenol extraction, chloroform extraction and ethanol precipitation. Approx. 250 ng DNA could be obtained from one individual .
  • the 50 ul reaction volume contained 0.5 ul of the digestion/ligation reaction as template as well as 10 pMol of an 5--FITC labelled primer complementary to the Xba adapter and having three selective nucleotides on its 3 ' end (CCG ;
  • SEQ ID NO 1 10 pMol of an unlabeled primer complementary to the BamHl adapter (no selective nucleotides ; SEQ ID NO 2) , 15 mM Tris-HCl pH 8.5 ; 50 mM KCl ; 1.5 mM MgC12 ; 0.01 % non-ionic detergent such as Triton X-100 ; 200 uM of each of the four dNTP ' s and 0.5 unit Taq polymerase.
  • the PCR mixture was overlayered with mineral oil.
  • Genomic DNA was isolated from L. esculentum (lane 1) and L. penelli (lane 2) and from 4 different individuals of a cross between these two species (lanes 3-6) .
  • Samples containing 20 ng samples DNA were processed as in example 4.
  • the following restriction-enzymes, adapters and PCR primers were used :
  • Restriction enzymes Xba I, BamHl and Rsa I.
  • Adapters Xba (SEQ ID NO 12 and SEQ ID NO 13) + Bam ( SEQ ID NO 9 and SEQ
  • This example pertains to the generation of DNA fingerprints from human genomic DNA (Promega) .
  • DNA was processed as in example 4. The same restriction- enzymes, adapters and PCR primers were used. The results are displayed in Fig. 5 and 6. In figure 5, the fragments having lengths between 65 and 710 nucleotides are displayed. Figure 6 shows a part of this same gel pattern.
  • the technique of the invention used for the mapping of transposons.
  • DNA- adapters of known sequence are ligated to the DNA ends and the DNA is denatured.
  • Tagged or immobilised oligonucleotides complementary to part of the transposon sequence are allowed to hybridise to the transposon containing fragments. DNA fragments not hybridised to tagged or immobilised oligonucleotides are removed.
  • the resulting DNA preparation which is strongly enriched in DNA fragments containing transposons is used as a template in an amplification reaction such as PCR using one primer complementary to the adapters ligated to the DNA fragments and one primer complementary to the transposon sequence.
  • the resulting amplification products are analysed.
  • the organism studied has more than 50 identical transposons in its genome it may be necessary to limit the number of amplified products by addition of one or more selective nucleotides to the primer complementary to the adapter-sequence .
  • the DNA fragment containing both transposon-sequences as well as sequences of the gene of interest can be identified and used for the characterisation of the gene of interest.
  • the technique of the invention used for the detection of polymorphisms.
  • the technique of the invention can be used to detect polymorphisms between the DNA-sequence of a small part of a genome and the homologous part of another genome .
  • Genomic DNA is digested with restriction-endonucleases to fragments of 4 - 40 Kb and the DNA is denatured.
  • Tagged or immobilised oligonucleotides complementary to part of the DNA fragment of interest are allowed to hybridise to the DNA-sample. DNA fragments not hybridised to tagged or immobilised oligonucleotides are removed.
  • the resulting single stranded DNA preparation which is strongly enriched in the DNA fragment of interest is made double-stranded using an oligonucleotide complementary to a sequence. at the end of the DNA molecule , a DNA polymerase and a suitable buffer containing deoxyribonucleotides .
  • the oligonucleotide used as a primer for the polymerase should be unique for the DNA fragment of interest .
  • the double stranded DNA preparation obtained is digested to small fragments with one or more restriction-endonucleases. Adapters are ligated to the DNA-ends and the resulting preparation of DNA fragments is amplified using oligonucleotides complementary to the adapters used.
  • the amplified fragments can be analysed directly, or after mixing with a control DNA preparation and one cycle of denaturation and renaturation by well known techniques that are not only sensitive to the length of the fragments obtained but also for the sequence of the DNA fragments, such as denaturing gradient gel electrophoresis (DGGE) and SSCP.
  • DGGE denaturing gradient gel electrophoresis
  • SSCP sequence of the DNA fragments
  • the technique of the invention used for the detection of polymorphisms between the DNA-sequence of a small part of a genome and the homologous part of another genome.
  • Genomic DNA is digested with restriction-endonucleases to fragments of 4 - 40 Kb.
  • Adapters are ligated to the DNA ends obtained and the DNA is denatured.
  • Tagged or immobilised oligonucleotides complementary to part of the DNA fragment of interest are allowed to hybridise to the DNA-sample. DNA fragments not hybridised to tagged or immobilised oligonucleotides are removed. The resulting single stranded DNA preparation which is strongly enriched in the DNA fragment of interest is used as a template in a DNA extension assay.
  • the DNA can be used directly in an extension assay or be amplified first e.g. with the use of the Polymerase Chain Reaction.
  • each hybridised oligonucleotide acts as a primer. that is elongated by Klenow fragment of E.coli polymerase I in the presence of deoxyribonucleotides and using the right conditions of buffer-composition and temperature. Elongation of each primer halts when the next hybridised primer is reached. After inactivation of the polymerase and denaturation of the DNA, the separate labelled fragments are analysed by techniques such as denaturing gradient gel electrophoresis and the patterns produced are compared with a control sample.
  • the primers used for the elongation reaction are chosen such that the fragments produced each have a different length and/or behaviour on the gels used for analysis.
  • the primers used for the elongation reaction are labelled.
  • the deoxyribonucleotides used for the elongation reaction are labelled.
  • the technique of the invention used to analyse a specific mRNA.
  • RNA preparation is incubated with a tagged or immobilised oligonucleotide or cDNA fragment that hybridises to the mRNA of interest. Non-hybridised RNA molecules are removed, the resulting RNA preparation which is strongly enriched for the mRNA of interest is used in an elongation assay as described above except that the DNA polymerase is replaced by a reverse transcriptase .
  • AFLP sequence tagging was used for the specific amplification of a single AFLP marker (Pstl/Msel P13/M61-385) in maize.
  • DNA of the parents of a recombinant inbred lines (RIL) population was used as starting material. These parentslines are named Mol7 and B73.
  • AFLP restriction/iigation mixes R/L mixes
  • PCR reactions were carried out using primercombinations Forward 11 and P13 and using 20-fold diluted +2/+3 AFLP reaction mixture from both parental lines as PCR template. The following PCR amplification profile was used: 30 cycles of 30 sec at 94°C; 60 seconds at 56°C and 60 seconds 72°C.
  • the PCR reaction mixture consisted of :
  • the DNA to be analysed can be genomic DNA from any organism including viruses, micro-organisms, plants, animals and human, but can also be cDNA made by reverse transcription from RNA.

Abstract

La présente invention concerne une méthode d'analyse d'acides nucléiques, qui consiste à fragmenter de l'ADN pour obtenir un groupe de fragments ayant au moins trois types différents de terminaisons, à ligaturer au moins deux petits fragments d'ADN (adaptateurs) aux différents types de terminaisons, à obtenir au moins une première amorce oligomère complémentaire du premier adaptateur et une seconde amorce oligomère complémentaire du second adaptateur, à amplifier lesdits fragments d'ADN, au moins un type de terminaison d'ADN ne prenant pas part à la réaction d'amplification, et à analyser le produit amplifié.
PCT/NL1999/000643 1998-10-16 1999-10-18 Procede de production d'empreintes genetiques WO2000023620A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU63720/99A AU6372099A (en) 1998-10-16 1999-10-18 Method for the generation of dna fingerprints

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP98203481 1998-10-16
EP98203481.1 1998-10-16

Publications (1)

Publication Number Publication Date
WO2000023620A1 true WO2000023620A1 (fr) 2000-04-27

Family

ID=8234225

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/NL1999/000643 WO2000023620A1 (fr) 1998-10-16 1999-10-18 Procede de production d'empreintes genetiques

Country Status (2)

Country Link
AU (1) AU6372099A (fr)
WO (1) WO2000023620A1 (fr)

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002083943A2 (fr) * 2001-04-12 2002-10-24 Epigenomics Ag Procede a microreseau permettant d'enrichir des fragments d'adn provenant de melanges complexes
EP1350853A1 (fr) * 2002-04-05 2003-10-08 ID-Lelystad, Instituut voor Dierhouderij en Diergezondheid B.V. Détection des polymorphismes
WO2007055568A1 (fr) * 2005-11-14 2007-05-18 Keygene N.V. Procede de tri a haut debit de populations de marquage de transposons et d'identification a grande echelle de sequences paralleles de sites d'insertion
US7741463B2 (en) 2005-11-01 2010-06-22 Illumina Cambridge Limited Method of preparing libraries of template polynucleotides
EP2363504A1 (fr) 2005-12-22 2011-09-07 Keygene N.V. Procédé à base AFLP pour la détection de polymorphismes à haut rendement
US8168388B2 (en) 2005-11-25 2012-05-01 Illumina Cambridge Ltd Preparation of nucleic acid templates for solid phase amplification
EP2694682A1 (fr) * 2011-04-05 2014-02-12 Dow AgroSciences LLC Analyse à haut débit de bordures de transgènes
US9328378B2 (en) 2006-07-31 2016-05-03 Illumina Cambridge Limited Method of library preparation avoiding the formation of adaptor dimers
US9388459B2 (en) 2002-06-17 2016-07-12 Affymetrix, Inc. Methods for genotyping
US9512478B2 (en) 2007-02-02 2016-12-06 Illumina Cambridge Limited Methods for indexing samples and sequencing multiple polynucleotide templates
EP3541950A4 (fr) * 2016-11-16 2020-06-03 Progenity, Inc. Dosage multimodal pour la détection d'aberrations de l'acide nucléique
US20210292812A1 (en) * 2010-06-21 2021-09-23 Life Technologies Corporation Compositions, methods and kits for nucleic acid synthesis and amplification
CN113539373A (zh) * 2021-06-28 2021-10-22 福建农林大学 一种dna样品中不同长度dna的条数分布的分析方法
US11306351B2 (en) 2005-12-21 2022-04-19 Affymetrix, Inc. Methods for genotyping

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2295011A (en) * 1994-11-10 1996-05-15 Unilever Plc Selection of DNA markers using adaptor molecules
GB2295228A (en) * 1994-11-10 1996-05-22 Unilever Plc Selective amplification of DNA having a plurality of sites for a specific endonuclease
WO1996017082A2 (fr) * 1994-11-28 1996-06-06 E.I. Du Pont De Nemours And Company Sondes de microsatellites composes pour la detection de polymorphismes genetiques
WO1998008981A1 (fr) * 1996-08-30 1998-03-05 Life Technologies, Inc. PROCEDES D'IDENTIFICATION ET D'ISOLEMENT DE SEQUENCES DE NUCLEOTIDES SPECIFIQUES DANS L'ADNc ET L'ADN GENOMIQUE
WO1998030721A1 (fr) * 1997-01-10 1998-07-16 Pioneer Hi-Bred International, Inc. Amplification et analyse genetiques sur la base d'une hybridation

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2295011A (en) * 1994-11-10 1996-05-15 Unilever Plc Selection of DNA markers using adaptor molecules
GB2295228A (en) * 1994-11-10 1996-05-22 Unilever Plc Selective amplification of DNA having a plurality of sites for a specific endonuclease
WO1996017082A2 (fr) * 1994-11-28 1996-06-06 E.I. Du Pont De Nemours And Company Sondes de microsatellites composes pour la detection de polymorphismes genetiques
WO1998008981A1 (fr) * 1996-08-30 1998-03-05 Life Technologies, Inc. PROCEDES D'IDENTIFICATION ET D'ISOLEMENT DE SEQUENCES DE NUCLEOTIDES SPECIFIQUES DANS L'ADNc ET L'ADN GENOMIQUE
WO1998030721A1 (fr) * 1997-01-10 1998-07-16 Pioneer Hi-Bred International, Inc. Amplification et analyse genetiques sur la base d'une hybridation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JANSSEN P ET AL: "Evaluation of the DNA fingerprinting method AFLP as a new tool in bacterial taxonomy", MICROBIOLOGY, vol. 142, no. 7, July 1996 (1996-07-01), pages 1881-93, XP002097675 *
SIMONS G ET AL: "AFLP-BASED FINE MAPPING OF THE MLO GENE TO A 30-KB DNA SEGMENT OF THE BARLEY GENOME", GENOMICS, vol. 44, no. 1, January 1997 (1997-01-01), pages 64 - 70, XP002049472 *

Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002083943A3 (fr) * 2001-04-12 2003-10-30 Epigenomics Ag Procede a microreseau permettant d'enrichir des fragments d'adn provenant de melanges complexes
WO2002083943A2 (fr) * 2001-04-12 2002-10-24 Epigenomics Ag Procede a microreseau permettant d'enrichir des fragments d'adn provenant de melanges complexes
EP1350853A1 (fr) * 2002-04-05 2003-10-08 ID-Lelystad, Instituut voor Dierhouderij en Diergezondheid B.V. Détection des polymorphismes
WO2003087409A1 (fr) * 2002-04-05 2003-10-23 Id-Lelystad, Instituut Voor Dierhouderij En Diergezondheid B.V. Detection de polymorphismes
US9388459B2 (en) 2002-06-17 2016-07-12 Affymetrix, Inc. Methods for genotyping
US10253359B2 (en) 2005-11-01 2019-04-09 Illumina Cambridge Limited Method of preparing libraries of template polynucleotides
US7741463B2 (en) 2005-11-01 2010-06-22 Illumina Cambridge Limited Method of preparing libraries of template polynucleotides
US9376678B2 (en) 2005-11-01 2016-06-28 Illumina Cambridge Limited Method of preparing libraries of template polynucleotides
US8563478B2 (en) 2005-11-01 2013-10-22 Illumina Cambridge Limited Method of preparing libraries of template polynucleotides
US11142789B2 (en) 2005-11-01 2021-10-12 Illumina Cambridge Limited Method of preparing libraries of template polynucleotides
WO2007055568A1 (fr) * 2005-11-14 2007-05-18 Keygene N.V. Procede de tri a haut debit de populations de marquage de transposons et d'identification a grande echelle de sequences paralleles de sites d'insertion
US8071310B2 (en) 2005-11-14 2011-12-06 Keygene Nv Method for the high throughput screening of transposon tagging populations and massive parallel sequence identification of insertion sites
US8168388B2 (en) 2005-11-25 2012-05-01 Illumina Cambridge Ltd Preparation of nucleic acid templates for solid phase amplification
US11306351B2 (en) 2005-12-21 2022-04-19 Affymetrix, Inc. Methods for genotyping
EP2789696A1 (fr) 2005-12-22 2014-10-15 Keygene N.V. Procédé pour la détection de polymorphisme à base AFLP à haut rendement
EP3404114A1 (fr) 2005-12-22 2018-11-21 Keygene N.V. Procédé pour la détection de polymorphisme à base aflp à haut rendement
EP2363504A1 (fr) 2005-12-22 2011-09-07 Keygene N.V. Procédé à base AFLP pour la détection de polymorphismes à haut rendement
EP3045544A1 (fr) 2005-12-22 2016-07-20 Keygene N.V. Procédé pour la détection de polymorphisme à haut rendement par aflp
US9328378B2 (en) 2006-07-31 2016-05-03 Illumina Cambridge Limited Method of library preparation avoiding the formation of adaptor dimers
US9512478B2 (en) 2007-02-02 2016-12-06 Illumina Cambridge Limited Methods for indexing samples and sequencing multiple polynucleotide templates
US10457985B2 (en) 2007-02-02 2019-10-29 Illumina Cambridge Limited Methods for indexing samples and sequencing multiple polynucleotide templates
US10988806B2 (en) 2007-02-02 2021-04-27 Illumina Cambridge Limited Methods for indexing samples and sequencing multiple polynucleotide templates
US11634768B2 (en) 2007-02-02 2023-04-25 Illumina Cambridge Limited Methods for indexing samples and sequencing multiple polynucleotide templates
US20210292812A1 (en) * 2010-06-21 2021-09-23 Life Technologies Corporation Compositions, methods and kits for nucleic acid synthesis and amplification
EP2694682A4 (fr) * 2011-04-05 2014-12-31 Dow Agrosciences Llc Analyse à haut débit de bordures de transgènes
JP2014511695A (ja) * 2011-04-05 2014-05-19 ダウ アグロサイエンシィズ エルエルシー トランスジーン境界の高スループット分析
EP2694682A1 (fr) * 2011-04-05 2014-02-12 Dow AgroSciences LLC Analyse à haut débit de bordures de transgènes
EP3541950A4 (fr) * 2016-11-16 2020-06-03 Progenity, Inc. Dosage multimodal pour la détection d'aberrations de l'acide nucléique
CN113539373A (zh) * 2021-06-28 2021-10-22 福建农林大学 一种dna样品中不同长度dna的条数分布的分析方法

Also Published As

Publication number Publication date
AU6372099A (en) 2000-05-08

Similar Documents

Publication Publication Date Title
EP0534858B2 (fr) Amplification sélective des fragments de restriction: procédé général pour le "fingerprinting" d'ADN
US5955276A (en) Compound microsatellite primers for the detection of genetic polymorphisms
US6027894A (en) Nucleic acid adapters containing a type IIs restriction site and methods of using the same
US5952176A (en) Glycosylase mediated detection of nucleotide sequences at candidate loci
AU753661B2 (en) A method for the characterisation of nucleic acid molecules involving generation of extendible upstream DNA fragments resulting from the cleavage of nucleic acid at an abasic site
US7846693B2 (en) Nucleic acid detection assay
US7935488B2 (en) Selective restriction fragment amplification: fingerprinting
CN105154556B (zh) 实时荧光恒温指数扩增方法
WO2000023620A1 (fr) Procede de production d'empreintes genetiques
JP5336350B2 (ja) Ｄｎａフラグメントの増幅
JPS63500006A (ja) エキソヌクレア−ゼ阻害による核酸の塩基配列決定法
AU652548B2 (en) Amplification methods
CN111100908B (zh) 一种检测核苷酸片段缺失的方法和试剂盒
US7026115B1 (en) Selective restriction fragment amplification: fingerprinting
JP2003510011A (ja) カップル性ポリメラーゼ連鎖反応−制限エンドヌクレアーゼ消化−リガーゼ検出反応法
CN113046421A (zh) 一种不对称扩增靶核酸的方法
CN113046420A (zh) 一种不对称扩增多个靶核酸的方法
CN114686574A (zh) 一种检测核苷酸大片段缺失的方法和试剂盒
AU2004270756B2 (en) Nucleic acid detection assay
CN117701557A (zh) 基于熔解曲线分析的核酸多重检测方法、探针组及试剂盒
CA2203143A1 (fr) Sondes de microsatellites composes pour la detection de polymorphismes genetiques
MXPA00010281A (en) A method for the characterisation of nucleic acid molecules involving generation of extendible upstream dna fragments resulting from the cleavage of nucleic acid at an abasic site

Legal Events

Date Code Title Description
ENP Entry into the national phase

Ref country code: AU

Ref document number: 1999 63720

Kind code of ref document: A

Format of ref document f/p: F

AK Designated states

Kind code of ref document: A1

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase